Biocidal compositions and use thereof containing a synergistic mixture of 2-bromo-2-nitropropane-1,3-diol and beta-bromo-beta-nitrostyrene

ABSTRACT

A bactericidal composition and method for inhibiting and controlling the growth of the capsulated, facultative bacterium, Klebsiella pneumoniae, are disclosed. The composition comprises an amount, effective for the intended purpose, of 2-bromo-2-nitropropane-1,3-diol (&#34;BNPD&#34;) and beta-bromo-beta-nitrostyrene (&#34;BNS&#34;). The method comprises administering between about 0.1 to about 200 parts of this combined treatment (based on one million parts of the desired aqueous system) to the particular water containing system for which treatment is desired.

BACKGROUND OF THE INVENTION

The formation of slimes by microorganisms is a problem that isencountered in many aqueous systems. For example, the problem is notonly found in natural waters such as lagoons, lakes, ponds, etc., andconfined waters as in pools, but also in such industrial systems ascooling water systems, air washer systems and pulp and paper millsystems. All possess conditions which are conducive to the growth andreproduction of slime-forming microorganisms. In both once-through andrecirculating cooling systems, for example, which employ largequantities of water as a cooling medium, the formation of slime bymicroorganisms is an extensive and constant problem.

Airborne organisms are readily entrained in the water from coolingtowers and find this warm medium an ideal environment for growth andmultiplication. Aerobic and heliotropic organisms flourish on the towerproper while other organisms colonize and grow in such areas as thetower sump and the piping and passages of the cooling system. The slimeformation not only aids in the deterioration of the tower structure inthe case of wooden towers, but also promotes corrosion when it depositson metal surfaces. Slime carried through the cooling system plugs andfouls lines, valves, strainers, etc., and deposits on heat exchangesurfaces In the latter case, the impedance of heat transfer can greatlyreduce the efficiency of the cooling system.

In pulp and paper mill systems, slime formed by microorganisms iscommonly encountered and causes fouling, plugging, or corrosion of thesystem. The slime also becomes entrained in the paper produced to causebreakouts on the paper machines, which results in work stoppages and theloss of production time. The slime is also responsible for unsightlyblemishes in the final product, which result in rejects and wastedoutput.

The previously discussed problems have resulted in the extensiveutilization of biocides in cooling water and pulp and paper millsystems. Materials which have enjoyed widespread use in suchapplications include chlorine, chlorinated phenols, organo-bromines, andvarious organo-sulfur compounds. All of these compounds are generallyuseful for this purpose but each is attended by a variety ofimpediments. For example, chlorination is limited both by its specifictoxicity for slime-forming organisms at economic levels and by thetendency of chlorine to react, which results in the expenditure of thechlorine before its full biocidal function is achieved. Other biocidesare attended by odor problems and hazards in respect to storage, use orhandling which limit their utility. To date, no one compound or type ofcompound has achieved a clearly established predominance in respect tothe applications discussed Likewise, lagoons, ponds, lakes, and evenpools, either used for pleasure purposes or used for industrial purposesfor the disposal and storage of industrial wastes, become, during thewarm weather, besieged by slime due to microorganisms growth andreproduction. In the case of the recreational areas the problem ofinfection is obvious. In the case of industrial storage or disposal ofindustrial materials, the microorganisms cause additional problems whichmust be eliminated prior to the material's use or disposal of the waste.

Naturally, economy is a major consideration in respect to all of thesebiocides Such economic considerations attach to both the cost of thebiocide and the expense of its application. The cost performance indexof any biocide is derived from the basic cost of the material, itseffectiveness per unit of weight, the duration of its biocidal orbiostatic effect in the system treated, and the ease and frequency ofits addition to the system treated. To date, none of the commerciallyavailable biocides has exhibited a prolonged biocidal effect. Instead,their effectiveness is rapidly reduced as the result of exposure tophysical conditions such as temperature, association with ingredientscontained by the system toward which they exhibit an affinity orsubstantivity, etc., with a resultant restriction or elimination oftheir biocidal effectiveness, or by dilution.

As a consequence, the use of such biocides involves their continuous orfrequent addition to systems to be treated and their addition tomultiple points or zones in the systems to be treated. Accordingly, thecost of the biocide and the labor cost of such means of applying it areconsiderable. In other instances, the difficulty of access to the zonein which slime formation is experienced precludes the effective use of abiocide. For example, if in a particular system there is no access to anarea at which slime formation occurs the biocide can only be applied ata point which is upstream in the flow system. However, the physical orchemical conditions, e.g., chemical reactivity, thermal degradation,etc., which exist between the point at which the biocide may be added tothe system and the point at which its biocidal effect is desired renderthe effective use of a biocide impossible.

Similarly, in a system experiencing relatively slow flow, such as apaper mill, if a biocide is added at the beginning of the system, itsbiocidal effect may be completely dissipated before it has reached allof the points at which this effect is desired or required. As aconsequence, the biocide must be added at multiple points, and even thena diminishing biocidal effect will be experienced between one point ofaddition to the system and the next point downstream at which thebiocides may be added. In addition to the increased cost of utilizingand maintaining multiple feed points, gross ineconomies in respect tothe cost of the biocide are experienced. Specifically, at each point ofaddition, an excess of the biocide is added to the system in order tocompensate for that portion of the biocide which will be expended inreacting with other constituents present in the system or experiencephysical changes which impair its biocidal activity.

SUMMARY OF THE INVENTION

The biocidal compositions of the present invention comprise, as activeingredients, (1) 2-bromo-2-nitropropane-1,3-diol ("BNPD") and (2)beta-bromo-beta-nitrostyrene ("BNS").

PRIOR ART

BNPD is sold by the Boots Company Ltd., Nottingham, England as anindustrial water treatment antibacterial agent. Of interest regardingthe uses of BNPD are Chemical Abstract, Volume 95 (1981) 199029f,disclosing the use of a composition comprising4,5-dichloro-1,2-dithiol-3-one and BNPD, and Bryce, et al. ChemicalAbstract, Volume 89 (1978) 117510 v, reviewing references describing themicrobial, chemical, analytical properties, and formulation of Bronopol(a trade name of Boots for BNPD). Neither one of the abstracts disclosesthe composition of the present invention nor the utilization of such acomposition to inhibit Klebsiella pneumoniae growth.

BNPD has been used in conjunction with other biocidal compounds asdescribed in the following U.S. patents of common assignment andinventorship herewith: U.S. Pat. No. 4,725,587 to Whitekettle andDonofrio, U.S. Pat. No. 4,725,623 to Whitekettle and Donofrio, U.S. Pat.No. 4,725,624 to Whitekettle and Donofrio, U.S. Pat. No. 4,732,905 toDonofrio and Whitekettle, U.S. Pat. No. 4,732,911 to Whitekettle andDonofrio, U.S. Pat. No. 4,732,913 to Donofrio and Whitekettle and U.S.Pat. No. 4,753,961 to Donofrio and Whitekettle.

BNS is a known biocidal composition available from Givaudan Corporation,New York, N.Y. Of interest regarding the uses of BNS are U.S. Pat. No.3,524,812 to Shema, U.S. Pat. No. 4,561,983 to Davis et al. and U.S.Pat. No. 4,579,665 to Davis et al. Although both BNPD and BNS are knownbiocidal compounds, the synergistic effect obtained by combining BNPDand BNS has not been previously disclosed.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the present inventors have found that mixtures of BNPD andBNS are especially efficacious in controlling the growth of bacterialmicrobes, specifically the Klebsiella pneumoniae species. Thisparticular species is a member of the capsulated, facultative class ofbacteria and is generally present in air, water and soil. These bacteriacontinually contaminate open cooling systems and pulping and papermakingsystems and are among the most common slime formers. The slime may beviewed as being a mass of agglomerated cells stuck together by thecementing action of the gelatinous polysaccharide or proteinaceoussecretions around each cell. The slimy mass entraps other debris,restricts water flow and heat transfer, and may serve as a site forcorrosion.

The fact that the Klebsiella species used in the tests is a facultativespecies is important as, by definition, such bacteria may thrive undereither aerobic or anaerobic conditions. Accordingly, by reason ofdemonstrated efficacy in the growth inhibition of this particularspecies, one can expect similar growth inhibition attributes when otheraerobic or anaerobic bacterial species are encountered. It is alsoexpected that these compositions will exhibit similar growth inhibitionattributes when fungi and algae species are encountered.

As noted above, BNPD is available from the Boots Company, Ltd. and issold under the trademarks "Myacide AS" or "Bronopol". It is a white,free flowing, crystalline solid that is readily soluble in cold water.The product is from about 95-100% pure.

BNS is available from Givaudan Corporation under the tradename Giv-GardBNS 25% AF. The pure compound is a white powder that is insoluble inwater and hydrolyzes quickly. BNS is soluble in dimethylformamide andhighly aromatic naphtha. The BNS used in the present invention is about98-100% pure.

In accordance with the present invention, the combined BNPD and BNStreatment may be added to the desired aqueous system in need of biocidaltreatment, in an amount of from about 0.1 to about 200 parts of thecombined treatment to one million parts (by weight) of the aqueousmedium. Preferably, about 5 to about 50 parts of the combined treatmentper one million parts (by weight) of the aqueous medium is added.

The combined treatment is added, for example, to cooling water systems,paper and pulp mill systems, pools, ponds, lagoons, lakes, etc., tocontrol the formation of bacterial microorganisms, which may becontained by, or which may become entrained in, the system to betreated. It has been found that the BNPD and BNS compositions andmethods of utilization of the treatment are efficacious in controllingthe facultative bacterium, Klebsiella pneumoniae, which may populatethese systems. It is thought that the combined treatment composition andmethod of the present invention will also be efficacious in inhibitingand controlling all types of aerobic and anaerobic bacteria.

Surprisingly, it has been found that when the BNPD and BNS ingredientsare mixed, in certain instances, the resulting mixtures possess a higherdegree of bactericidal activity than that of the individual ingredientscomprising the mixture. Accordingly, it is possible to produce a highlyefficacious bactericide. Because of the enhanced activity of themixture, the total quantity of the bacterial treatment may be reduced.In addition, the high degree of bactericidal effectiveness which isprovided by each of the ingredients may be exploited without use ofhigher concentrations of each.

The following experimental data were developed. It is to be rememberedthat the following examples are to be regarded solely as beingillustrative, and not as restricting the scope of the invention.

DESCRIPTION OF PREFERRED EMBODIMENT

BNPD and BNS were added in varying ratios and over a wide range ofconcentrations to a liquid nutrient medium which was subsequentlyinoculated with a standard volume of a suspension of the facultativebacterium Klebsiella pneumoniae. Growth was measured by determining theamount of radioactivity accumulated by the cells when ¹⁴ C-glucose wasadded as the sole source of carbon in the nutrient medium. The effect ofthe biocide chemicals, alone and in combination, is to reduce the rateand amount of ¹⁴ C incorporation into the cells during incubation, ascompared to controls not treated with the chemicals. Additions of thebiocides, alone and in varying combinations and concentrations, weremade according to the accepted "checkerboard" technique described by M.T. Kelley and J. M. Matsen, Antimicrobial Agents and Chemotherapy. 9:440 (1976). Following a two hour incubation, the amount of radioactivityincorporated in the cells was determined by counting (¹⁴ C liquidscintillation procedures) for all treated and untreated samples. Thepercent reduction of each treated sample was calculated from therelationship: ##EQU1##

Plotting the % reduction of ¹⁴ C level against the concentration of eachbiocide acting alone results in a dose-response curve, from which thebiocide dose necessary to achieve any given % reduction can beinterpolated.

Synergism was determined by the method of calculation described by F. C.Kull, P. C. Eisman, H. D. Sylwestrowicz and R. L. Mayer, AppliedMicrobiology 9:538 (1961) using the relationship. ##EQU2## where: Q_(a)=quantity of compound A, acting alone, producing an end point

Q_(b) =quantity of compound B, acting alone, producing an end point

Q_(A) =quantity of compound A in mixture, producing an end point

Q_(B) =quantity of compound B in mixture, producing an end point

The end point used in the calculations is the % reduction caused by eachmixture of A and B. Q_(A) and Q_(B) are the individual concentrations inthe A/B mixture causing a given % reduction. Q_(a) and Q_(b) aredetermined by interpolation from the respective dose-response curves ofA and B as those concentrations of A and B acting alone which producethe same % reduction as each specific mixture produced.

Dose-response curves for each active acting alone were determined bylinear regression analysis of the dose-response data. After linearizingthe data, the contributions of each biocide component in the biocidemixtures to the inhibition of radioisotope uptake were determined byinterpolation with the dose-response curve of the respective biocide.If, for example, quantities of Q_(A) plus Q_(B) are sufficient to give a50% reduction in ¹⁴ C content, Q_(a) and Q_(b) are those quantities of Aor B acting alone, respectively, found to give 50% reduction in ¹⁴ Ccontent. A synergism index (SI) is calculated for each combination of Aand B.

Where the SI is<1, synergism exists. Where the SI=1, additivity exists.Where SI>1, antagonism exists.

The data in the following tables come from treating Klebsiellapneumoniae, a common nuisance bacterial type found in industrial coolingwaters and in pulping and paper making systems, with varying ratios andconcentrations of BNPD and BNS. Shown for each combination is the %reduction of ¹⁴ C content (% I), the calculated SI, and the weight ratioof BNPD to BNS.

                  TABLE I                                                         ______________________________________                                        BNPD vs. BNS                                                                  ppm       ppm    Ratio                                                        BNPD      BNS    BNPD:BNS     % I  SI                                         ______________________________________                                        2.5       0      100:0        0                                               5         0      100:0        0                                               10        0      100:0        0                                               20        0      100:0        14                                              40        0      100:0        76                                              80        0      100:0        91                                              0         1.25   0:100        0                                               0         2.5    0:100        0                                               0         5      0:100        0                                               0         10     0:100        77                                              0         20     0:100        89                                              0         40     0:100        94                                              2.5       40     1:16         94   1.28                                       5         40     1:8          94   1.32                                       10        40     1:4          95   1.35                                       20        40     1:2          95   1.48                                       40        40     1:1          86   2.19                                       80        40     2:1          92   2.45                                       2.5       20     1:8          90   0.75*                                      5         20     1:4          90   0.78*                                      10        20     1:2          92   0.80*                                      20        20     1:1          93   0.92*                                      40        20     2:1          93   1.19                                       80        20     4:1          95   1.67                                       2.5       10     1:4          80   0.52*                                      5         10     1:2          79   0.58*                                      10        10     1:1          86   0.55*                                      20        10     2:1          90   0.64*                                      40        10     4:1          94   0.92*                                      80        10     8:1          95   1.37                                       2.5       5      1:2          0                                               5         5      1:1          24   1.48                                       10        5      2:1          56   0.72*                                      20        5      4:1          77   0.59*                                      40        5      8:1          86   0.92*                                      80        5      16:1         93   1.26                                       2.5       2.5    1:1          0                                               5         2.5    2:1          0                                               10        2.5    4:1          4    2.68                                       20        2.5    8:1          55   0.69*                                      40        2.5    16:1         87   1.14                                       80        2.5    32:1         94   1.16                                       2.5       1.25   2:1          0                                               5         1.25   9:1          0                                               10        1.25   8:1          0                                               20        1.25   16.1         37   0.85*                                      40        1.25   32:1         84   1.50                                       80        1.25   64:1         94   1.12                                       ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        BNPD vs. BNS                                                                  ppm       ppm    Ratio                                                        BNPD      BNS    BNPD:BNS     % I  SI                                         ______________________________________                                        2.5       0      100:0        0                                               5         0      100:0        0                                               10        0      100:0        0                                               20        0      100:0        20                                              40        0      100:0        79                                              80        0      100:0        95                                              0         1.25   0:100        0                                               0         2.5    0:100        0                                               0         5      0:100        21                                              0         10     0:100        81                                              0         20     0:100        92                                              0         40     0:100        96                                              2.5       40     1:16         96   1.41                                       5         40     1:8          96   1.45                                       10        40     1:4          96   1.52                                       20        40     1:2          96   1.66                                       40        40     1:1          96   1.93                                       80        40     2:1          97   2.43                                       2.5       20     1:8          92   0.81*                                      5         20     1:4          94   0.81*                                      10        20     1:2          95   0.85*                                      20        20     1:1          95   0.99                                       40        20     2:1          95   1.27                                       80        20     4:1          97   1.76                                       2.5       10     1:4          83   0.54*                                      5         10     1:2          86   0.69*                                      10        10     1:1          87   0.60*                                      20        10     2:1          92   0.67*                                      40        10     4:1          94   0.93*                                      80        10     8:1          97   1.43                                       2.5       5      1:2          28   1.36                                       5         5      1:1          49   0.85*                                      10        5      2:1          66   0.61*                                      20        5      4:1          84   0.50*                                      40        5      8:1          94   0.75*                                      80        5      16:1         97   1.26                                       2.5       2.5    1:1          0                                               5         2.5    2:1          0                                               10        2.5    4:1          20   1.42                                       20        2.5    8:1          65   0.61*                                      40        2.5    16:1         90   0.69*                                      80        2.5    32:1         96   1.20                                       2.5       1.25   2:1          0                                               5         1.25   4:1          0                                               10        1.25   8:1          0                                               20        1.25   16:1         51   0.66*                                      40        1.25   32:1         88   0.65*                                      80        1.25   64:1         95   1.16                                       ______________________________________                                    

Asterisks in the SI column indicate synergistic combinations inaccordance with the Kull method supra.

In Tables I and II, differences seen between the replicates are due tonormal experimental variance.

In accordance with Tables I-II supra., unexpected results occurred morefrequently within the product ratios of BNPD to BNS of from about 32:1to 1:8. At present, it is preferred that the commercial productembodying the invention comprise a weight ratio of about 1:1 BNPD:BNS.

While this invention has been described with respect to particularembodiments thereof, it is apparent that numerous other forms andmodifications of this invention will be obvious to those skilled in theart. The appended claims and this invention generally should beconstrued to cover all such obvious forms and modifications which arewithin the true spirit and scope of the present invention.

We claim:
 1. A composition comprising a synergistic aqueous mixture of(a) 2-bromo-2-nitropropane-1,3 diol (BNPD) and (b)beta-bromo-beta-nitrostyrene (BNS), wherein the weight ratio of saidBNPD to said BNS is from about 32:1 to 1:8.
 2. The composition asrecited in claim 1 wherein the weight ratio of said BNPD to said BNS is1:1.
 3. A method for controlling the growth of Klebsiella pneumoniaebacteria in an aqueous system which comprises adding to said system fromabout 0.1 to 200 parts per weight of a composition per one million partsper weight of said aqueous system, said composition comprising asynergistic aqueous mixture of (a) 2-bromo-2-nitropropane-1,3 diol(BNPD) and (b) beta-bromo-beta-nitrostyrene (BNS), the weight ratio ofsaid BNPD to said BNS being from about 32:1 to 1:8.
 4. The method asrecited in claim 3 wherein the weight ratio of BNPD:BNS is about 1:1. 5.The method as recited in claim 3 wherein said composition is added tosaid system in an amount of from about 5 to about 50 parts per millionof said aqueous system.
 6. The method as recited in claim 3 wherein saidaqueous system comprises a cooling water system.
 7. The method asrecited in claim 3 wherein said aqueous system comprises a pulping andpapermaking system.